Summary: The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self-renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We have shown that Hmgb3 is a protein the binds both to transcription factors and to chromatin and that Hmgb3 deficiency activates the Wnt signalling pathway, leading to stem cell division and the balance between self-renewal and differentiation. We hypothesize that Wnt antagonists regulate this balance in vivo and are developing tools to manipulate Wnt expression by stromal cells and hematopoietic cells in vivo. Specific Aim 1.2: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. We are extending our studies of Wnt signaling to muscle stem cells to determine whether this population of stem cells is also regulated by Wnt signaling. Project 2: We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease and the adverse events in other gene therapy trials point out the problem of inserting powerful enhancers like those from the globin locus into the genome. Specific aim 2.1: We have shown that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells, and that this leads to improved gene transfer to human hematopoietic cells in the sheep xenograft model. We are adapting the FeLV-C envelope to pseudotype lentivirus vectors to suppress globin vector instability and improve gene transfer frequency. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia without enhancer elements. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter directs the necessary level of globin but needs a barrier element for position independent, uniform, high-level, and copy number dependent expression. We demonstrated a compact insulator element in the ankyrin promoter that provides protection from gene silencing in vitro and in vivo, as well as barrier elements in the alpha spectrin and AHSP loci. We have generated vectors with combinations of barriers flanking the B3 globin gene and are evaluating these in mouse models. We have identified a TFIID binding region in the ankyrin promoter that we have modified to increase expression of reporter genes linked to this promoter. We are currently incorporating this promoter into vectors for analysis in mouse models.